Phosphofructokinase (PFK) is clearly established as the enzyme catalyzing one of the principal rate controlling steps of glycolysis in virtually all tissues. The mechanism of regulation of this key pacemaker is a subject of continued interest, as mechanistic knowledge acquired in the study of this enzyme has general applicability to other allosteric enzymes. This continuing project will: (1) Study the allosteric regulatory properties and tissue distribution of phosphofructokinase-C, which is a major, unique isozyme of the brain. Among the properties to be examined are the kinetic consequence of protein phosphorylation, the regulatory role of fructose-2,6-P2, and the possible role of other regulatory ligands that may be important to the control of carbohydrate metabolism in the brain. As a part of these studies, monoclonal antibodies will be prepared against the A, B, and C isozymes of PFK. The antibodies will be used in isozyme isolation and in tissue distribution studies, and may provide tools for the isolation of homologous sequences among PFK isozymes. (2) Continue ongoing studies of the association of muscle PFK with F-actin, and to examine the role of PFK phosphorylation and the role of regulatory ligands on the binding phenomenon. (3) Extend studies of the tertiary structure and the multiple conformational forms of PFK by employing the technique of limited proteolysis coupled with primry structure analysis. (4) Study the kinetic consequences of the chemical modification of allosteric and catalytic sites, including modification of the AMP site with 2-azido-AMP, and modification of the ATP catalytic site with dialdehyde ATP, dialdehyde ITP, and 5[p-(fluorosulfonyl)benzoyl]adenosine. In each of these last instances the modified enzyme will be used in a parallel project to determine the complete covalent structure of PFK and to place these sites in the primary structure of the enzyme.